Tuberculosis remains a major global health problem and vaccine development is a critical priority. T-cells play a pivotal role in protection against Mycobacterium tuberculosis. However, M. tuberculosis is known to inhibit human immunity, but the mechanisms are not well understood. Early secreted antigenic target of 6 kD (ESAT-6) is an immunogenic protein that is a potential vaccine candidate, but also contributes to virulence and cytolysis of mammalian cells. Our preliminary data indicate that ESAT-6 directly inhibits human T cell proliferation and IFN-g secretion, and these effects are not due to cytotoxicity or increased apoptosis. Our goal is to understand the molecular mechanisms by which ESAT-6 inhibits human T cell IFN-g production. The specific aims are: 1. Identify the signaling pathways by which ESAT-6 inhibits T cell IFN-g production. We will determine the effect of ESAT-6 on transcription factors that bind to the IFN-g proximal promoter, using promoter pull-down assays and chromatin immunoprecipitation (aim 1.1). We will evaluate the effect of ESAT-6 on molecules that mediate signaling through the T-cell receptor (aim 1.2) and on the intermediary signaling molecules, mitogen-activated protein kinases (aim 1.3), using immunoprecipitation, Western blotting and functional kinase assays 2. Identify a mammalian ligand for ESAT-6 on human T-cells. We will use biotinylated ESAT-6 to pull down proteins from extracts of purified T-cells, elute the proteins, separate them by SDS-PAGE and identify them by mass spectrometry (aim 2.1). The putative ligand will be cloned and expressed, and interactions between ESAT-6 and its ligand will be confirmed by surface plasmon resonance and protein overlay assays (aim 2.2). To determine the functional significance of the interaction between ESAT-6 and its putative ligand, we will determine if neutralizing antibodies and/or siRNA to the ESAT-6 ligand abrogate the inhibitory effects of ESAT-6 on T-cells (aim 2.3). PUBLIC HEALTH RELEVANCE: Tuberculosis remains a tremendous public health problem world-wide, and an effective vaccine would contribute greatly to disease control. Early secreted antigenic target of 6 kD (ESAT-6) is a Mycobacterium tuberculosis protein that is a promising vaccine candidate, but also contributes to virulence and tissue damage. This proposal seeks to understand the mechanisms by which ESAT-6 inhibits human T-cell immune function, information that is critical for development of improved vaccines and treatment strategies for tuberculosis.